Understanding the process and anticipating the problems can help ensure successful separations.

The use of magnetic beads for capturing biological molecules has become commonplace in the life sciences. For in vitro applications, the beads coated with the right biomarker are incubated with the suspension containing the target. This can be an antigen, antibody, protein, cell, or nucleic acid. A magnetic force is applied and the magnetic beads captured, removing the supernatant with the rest of the sample. Clean buffer can be added to get the purified molecule resuspended if necessary.

So far, the most successful commercial application is chemiluminescence immunoassays (CLIA). The use of magnetic beads allows automation of the tests that make throughputs of thousands of tests by day possible. This helps hospital labs check 5–10 analytes per patient, which would be difficult using ELISAs or other older techniques. Since most technologies (coating, biomarkers, etc.) used in agglutination assays are similar, the transition from classical technologies to CLIA has been smooth. 

The industrialization of biomagnetic separation implies the need for better protocols, validation methods, and quality controls for all processes. Many protocols work fine at R&D laboratories that feature small volumes and manual operation. However, commercial robotic analyzers and large volume IVD production lines need stricter requirements. Even when working with small volumes, automatized analyzers require a well-controlled and repetitive separation time for each test. The production lines need to work with volumes large enough to cope with the demand generated by the robotic analyzers. Nonetheless, they also require a high degree of in-lot and lot-to-lot consistency to minimize the end-product variability.

For the past 10 years, we have helped companies improve their biomagnetic separation processes and instruments. This experience has helped us realize that most of the problems have the same root, which is the issue of biomagnetic separation conditions being unknown and only the separation time being fixed.

Biomagnetic Separation Working Conditions

A standard magnetic separation rack is an assembly of permanent magnets generating a magnetic force over the beads. What is the value of this force? If we look at physics’ textbooks, the magnetic force is the gradient of the product of the magnetic moment of the bead by the applied field. The bead’s magnetic moment depends on the value of the magnetic field. Far from the retention area—when the field is small—the beads have constant susceptibility and their magnetic moment will vary linearly with the applied field. Near the retention area, the magnetic field can be big enough to saturate the magnetic moment and the beads act like small permanent magnets.

In these conditions, we cannot define a single value for the magnetic force. Due to this, most of the magnetic separation processes are usually defined by the separation time only. Notice that this parameter is the consequence of the magnetic force applied and the distance traveled by the beads, it does not define the separation conditions. If you change the separator device, or the vessel’s geometry, the established separation time is no longer valid.

This is not the only problem. Depending on their position, the beads can experience a completely different force during the process; the ones placed farther would be under a very weak force during most of the process, while only the ones arriving to the retention area (if the separation time is large enough) would experience a strong force. By contrast, the beads placed near the retention area would experience a very strong force throughout process, forming irreversible aggregates (clumps). These different conditions for the beads, which mainly depend on their initial position, can lead to in-lot inconsistency and require dealing with large IVD-kit to kit variability.

If the separation time is shortened to avoid irreversible aggregation problems, many beads will not reach the retention area and they will be lost with the biomarkers and the captured target when the supernatant is extracted. If the separation time is increased to avoid losses, we will be contributing to the formation of clumps among the beads arriving early to the retention area.

A similar problem can be experienced when modifying the magnetic force at the retention area. If we try to reduce the retention force at long distance, the force will proportionally be much weaker, exponentially increasing separation time. If we try to have a stronger force, we will increase the retention force and thus the number and severity of the clumps.

Correctly Defining the Process

To define the biomagnetic separation process and then validate it, regardless of the specific device and volume, it is necessary to work on homogenous conditions. If all the beads experience the same magnetic force, in-lot consistency is guaranteed and the separation time is directly proportional to the distance traveled. If the same homogenous conditions are kept, we will be able to scale up the process just by proportionally increasing the separation time to the diameter of the new vessel.

Advanced biomagnetic separation systems successfully implemented this approach 10 years ago. To get a magnetic force, which does not depend on distance, we have to determine the value of the magnetic field that saturates the magnetic moment of the beads. With the beads saturated, we can apply a constant magnetic gradient and therefore obtain a constant force in all the working volume. Needless to say that, in order to succeed, we would have to carefully select the value of the magnetic force. The magnetic beads have to be retained with enough force so that they are not aspired when the supernatant is extracted. Nonetheless, the value should be gentler than any classical magnetic separation rack.

At the same time, even for the gentler retention force, the force at large distance would be much higher than an inhomogeneous magnetic force device. This would also shorten the separation time, allowing an earlier start of the supernatant extraction. By reducing the retention force and the time the beads remain in the retention area, we reduce the risk of irreversible aggregation.

Experience shows that with the right homogenous biomagnetic separation conditions it is not necessary to use sonication for re-suspending the beads, as no clumps are formed.

Lessons for R&D Labs and Small Volumes

Until now, large volume producers and automatized analyzers are the ones who have benefited the most from homogenous biomagnetic separation. Even if in many cases the described problems are not detected at R&D scale, it does not mean that these problems are nonexistent.

When a new CLIA IVD kit, or any other magnetic bead application, is developed, low performances are attributed to the magnetic bead, the coating protocol, or both. The usual reaction is to test new beads and/or providers, and other surfaces or protocols, which means investing more time and resources to redo the whole project. However, it is not unusual to also have poor results, similar to the ones before, in the second try.

Similar to using large volume vessels or limited separation time, the use of inhomogeneous magnetic separation racks can also cause poor performance. Many studies on reusability of magnetic bead for enzymatic catalysis or protein purification are stopped after a few steps because the bead losses are too high. Then, there is no way to determine whether the activity loss is because the enzyme/biomarker is degraded or because the number of beads has decreased.

It is best to fully comprehend the biomagnetic separation process itself first, so that, if during the development an issue is presented, the real cause can be identified. Although coating problems cannot always be disregarded, testing a new magnetic separation rack is faster and cheaper than redoing the project again.

Lluís M. Martínez, Ph.D., ([email protected]) is the CSO of Sepmag.

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